Viruses with segmented genomes, including pathogens such as influenza virus, Rotavirus and Bluetongue virus (BTV), face the collective challenge of packaging their genetic material in terms of the correct number and types of segments. Here we de- velop a novel network approach to predict RNA–RNA interactions between different genomic segments. Experimental data on RNA complex formation in the multi-segmented BTV genome are used to establish proof-of-concept of this technique. In particular, we show that trans interactions between segments occur at multiple specific sites, termed segment assortment signals (SASs) that are dispersed across each segment. In order to validate the putative trans acting networks, we used various biochemical and molecular techniques which confirmed predictions of the RNA network approach. A combination of mutagenesis and reverse genetics systems revealed that the RNA–RNA interacting sites identified are indeed responsible for segment assortment and complex formation, which are essential criteria for genome packaging. This paves the way for their exploitation as novel types of drug target, either to inhibit assembly, or for designing defective interfering particles containing an incomplete set of genomic segments.